US5420285A - Protein labelling utilizing certain pyridyl hydrazines, hydrazides and derivatives - Google Patents

Protein labelling utilizing certain pyridyl hydrazines, hydrazides and derivatives Download PDF

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US5420285A
US5420285A US08/026,426 US2642693A US5420285A US 5420285 A US5420285 A US 5420285A US 2642693 A US2642693 A US 2642693A US 5420285 A US5420285 A US 5420285A
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oxysuccinimidyl
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David A. Schwartz
Michael J. Abrams
Christen M. Giandomenico
Jon A. Zubieta
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    • C07D207/30Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
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    • A61K47/6889Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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    • C07D277/02Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D401/04Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07D401/12Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531Production of immunochemical test materials
    • G01N33/532Production of labelled immunochemicals
    • G01N33/534Production of labelled immunochemicals with radioactive label
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00Chemistry: analytical and immunological testing
    • Y10S436/804Radioisotope, e.g. radioimmunoassay

Abstract

Bifunctional aromatic compounds which are capable of linking metal ions to biologically useful molecules. The bifunctional compounds are characterized as having a hydrazine or hydrazide group and a protein reactive group. The hydrazine or hydrazide group may be protected as a lower alkyl hydrazone. Conjugates of the bifunctional compounds with macromolecules are also described and labelled macromolecules comprised of the conjugates and metal ions are provided. Additionally, a method is provided for forming a labelled macromolecule by reacting a conjugate with a metal species. The compounds and method of this invention are particularly useful in the fields of biology and medicine for imaging and/or therapy.

Description

This is a division of application Ser. No. 07/888,282, filed May 26, 1992, now U.S. Pat. No. 5,206,370, which is a continuation of Ser. No. 07/483,201, filed Feb. 21, 1990, now abandoned, which is a continuation-in-part of Ser. No. 07/315,270, filed Feb. 24, 1989, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to bifunctional compounds capable of linking metal ions, particularly technetium and rhenium, to biologically useful molecules.

Because of their high biological specificity, certain macromolecules (e.g., monoclonal antibodies) have been used to target radioisotopes to specific in vivo sites for the purpose of imaging and/or therapy. The use of the metastable isotope of technetium, 99m Tc, in diagnostic nuclear medicine is well established and the beta-emitting isotopes of rhenium 186 Re, 188 Re and 189 Re can be used therapeutically. A number of methods for attaching technetium to macromolecules have been described. Some of these methods involve the reduction of disulfide groups in the macromolecule (usually an immunoglobulin) to thiols and the subsequent use of these groups to bind reduced Tc (e.g., McKenzie et al., International Publication #WO 87/04164; and Bremer et al., EPO 271 806 A2). Methods of this type have several potential disadvantages. The reduction of disulfide units can lead to protein de-naturation and a subsequent loss in biological specificity. Also, the method cannot be used to label macromolecules lacking disulfide moieties.

Alternatively, 99m Tc can be linked to macromolecules via bifunctional chelates such as DTPA (D. Lanteigne and D. J. Hnatowich, Int. J. Appl. Radiat. Isot., Vol. 35(7), p. 617 (1984), chelating thiosemicarbazones (Y. Arano et al., Int. J. Nucl. Med. Biol., Vol. 12, p. 425 (1985), and diamide-dithiol ligands (A. Fritzberg, European Patent Appl. EP 188256 2A). Problems associated with these methods include significant nonspecific binding of technetium (binding to the protein at sites other than the chelating group) and slow kinetics of Tc-labelling.

Accordingly, it is the object of the present invention to provide new bifunctional molecules having hydrazine or hydrazide groups and protein reactive groups which can be used to link metal ions, such as 99m Tc, to macromolecules.

Another object of the present invention is to provide a method for labelling macromolecules with metal ions in which binding of the metal at sites other than the chelating group is minimal, and in which labelling occurs at a relatively fast rate (less than one hour at room temperature).

SUMMARY OF THE INVENTION

According to the invention, novel bifunctional hydrazine and hydrazide compounds, as well as conjugates thereof, are provided. Methods of labelling the conjugates with metal ions are also provided.

Broadly, the hydrazine and hydrazide compounds described herein as bifunctional aromatic hydrazines or hydrazides having a protein reactive substituent and a negative counterion. A modification of this invention is also provided in which the hydrazine or hydrazide function is protected as a lower alkyl hydrazone.

In another embodiment of the invention, conjugates are formed by reacting bifunctional hydrazine or hydrazide compounds of the invention with macromolecules such as proteins, polypeptides or glycoproteins. The bifunctional compounds react with nucleophilic groups on the macromolecules (e.g. lysine residues) to yield conjugates containing free hydrazine/hydrazide groups.

In a third embodiment, labelled macromolecules comprised of conjugates and metal ions are formed.

In a fourth embodiment, a method is provided for labelling macromolecules by reacting a conjugate of the invention with a metal species.

DETAILED DESCRIPTION OF THE INVENTION

The novel hydrazine and hydrazide compounds of the present invention are represented by one of the following formulas (I) or (II): ##STR1## wherein: A is a carbon or nitrogen atom;

B is a carbon or nitrogen atom;

D is a direct bond (to the 2-, 3-, or 4-position of the ring), CH2, C═O or ##STR2## E is C═O or together with F forms a maleimidyl group; F is a group readily replaced by a primary amine in neutral or basic aqueous media when E is C═O or together with E forms a maleimidyl group;

R is hydrogen or a lower alkyl group;

R' and R" may be the same or different and are selected from hydrogen and lower alkyl; and

X is a negative counterion.

Another embodiment of the invention includes compounds of the formula (III): ##STR3## where R, R', R", E and F have the values given above.

When E is carbonyl C═O, F is any group which, in combination with the attached carbonyl group, forms an active ester or active amide. Examples of suitable species for F include such diverse groups as N-oxysuccinimidyl, tetrafluorophenolate, N-oxybenztriazole and imidazolate. These examples are not intended to be construed as limiting the scope of the invention.

Suitable groups for R, R' and R" include, but are not limited to, the following: H, CH3, C2 H5, and C3 H7.

Examples of useful X ions are halides, nitrate, trifluoroacetate, tetrafluoroborate and sulfate. These examples are not intended to limit the scope of suitable counterions.

The above-described compounds are stable, isolable derivatives of molecules that contain two cross-reactive moieties: a hydrazine/hydrazide group and a protein reactive group such as an active ester, active amide or maleimido group.

In the synthesis of these stable derivatives, an acid labile protecting group such as t-butoxycarbonyl (t-BOC) is removed from the hydrazine/hydrazide under anhydrous acidic conditions, leaving the protein reactive group unchanged and the hydrazine/hydrazide group in an unreactive, protonated form. Alternatively, the hydrazine/hydrazide group can be protected as a lower alkyl hydrazone.

When a bifunctional compound having a protonated (or hydrazone protected) hydrazine/hydrazido function is then combined with a macromolecule such as a protein, polypeptide or glycoprotein in neutral or slightly basic media, preferably a pH of about 7-8.5, the protein-reactive part of the compound will react with nucleophilic groups on the protein, polypeptide or glycoprotein (e.g., amine groups such as lysine residues) to yield a conjugate containing free hydrazine/hydrazide groups. In the case of hydrazone conjugates, the free hydrazine/hydrazide is formed by dialysis into an acidic (pH 5.6) buffer. Because this type of conjugate includes a hydrazine or hydrazide, a strong metal binding group, it will then readily react when mixed with a suitable metal species in acidic media to yield a labelled protein, polypeptide or glycoprotein.

The metal species may be, for example, a reduced Tc species formed by reacting TcO4 - with a reducing agent, for example, stannous ion, in the presence of a chelating oxygen ligand (e.g. glucoheptonate). Examples of suitable reduced Tc species include Tc-glucoheptonate, Tc-gluconate, Tc-2-hydroxyisobutyrate, Tc-lactate and Tc-4,5-dihydroxy 1,3-benzene disulfonate. Other metals and ands are also within the scope of the invention.

A Tc labelling process can be conveniently performed in an aqueous buffer, preferably at a pH of about 4.5-6.5, in one hour or less. Reaction with other suitable metal species occurs in a similar manner under similar conditions.

Radiochemical yield as determined by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) using Tc is ≧90%. Treatment of protein with nonlinkable analogs, (i.e., compounds without a protein reactive carbonyl group, such as 4-hydrazinobenzoic acid or 6-hydrazinopyridine-3-carboxylic acid) does not yield protein capable of significant Tc binding, thus demonstrating the high specificity of this technique.

The technetium atoms are believed to be bound to the conjugate via a hydrazido or diazenido linkages: ##STR4##

Wherein: L is an ancillary dioxygen ligand. Examples of this type of linkage have been described for Mo and Re (Comprehensive Coordination Chemistry, Vol. 2, G. Wilkinson, ed., Pergamon (Oxford) 1987, p. 130-151) and several analogous complexes of 99 Tc have been prepared by the reaction of an organohydrazine derivative and a Tc(V) oxo species.

The above labelling scheme has en used to label polyclonal human IgG and the Fc region of human IgG. The Tc-conjugates have been used to image focal sites of infection in a rat model. The labelling scheme has also been used to label fragment E, (see L. C. Knight et al, J. Clin. Invest., Vol. 72, 1983, p. 2007-2013) which was used to image thrombi in a rabbit model for deep vein thrombosis and the monoclonal antibody 5E8.

EXAMPLES

The NMR and IR data given in the examples was obtained as follows:

1 H NMR spectra were recorded on an 80 MHz IBM AF-80 Spectrometer. All 1 H NMR results were recorded in DMSO-d6 unless otherwise indicated. IR spectra were recorded on a Perkin-Elmer 598 infrared spectrometer. NMR and IR spectra were consistent with assigned structure.

Compound names given in brackets below the title compounds in the various examples conform to Chemical Abstracts service index nomenclature. Reaction schemes are illustrated in the accompanying FIGS. 1-8 which correspond to the 7 drawing sheets. Each FIGURE shows the preparation of an individual compound according to the invention.

FIG. 1 illustrates the reaction for the preparation of succinimidyl 4-hydrazinobenzoate hydrochloride;

FIG. 2 illustrates the reaction for the preparation of succinimidyl 6-hydrazinopyridine-3-carboxylate hydrochloride;

FIG. 3 illustrates the reaction for the preparation of succinimidyl 4-hydrazidoterephthalate hydrochloride;

FIG. 4 illustrates the reaction for the preparation of 5-maleimidyl-2-hydrazinopyridine hydrochloride;

FIG. 5 illustrates the reaction for the preparation of succinimidyl 2-(2-propenylhydrazone)nicotinate;

FIG. 6 illustrates the reaction for the preparation of succinimidyl 2-(2-(1-propenyl)hydrazone))-thiazole-4-carboxylate;

FIG. 7 illustrates the reaction for the preparation of succinimidyl 2-(2-methylethenylhydrazone) thiazole-4-carboxylate; and

FIG. 8 illustrates the reaction for the preparation of succinimidyl 4-thiosemicarbazidobenzoate hemihydrochloride.

EXAMPLE 1 Preparation of Succinimidyl 4-hydrazinobenzoate Hydrochloride [2, 5-pyrrolidinedione, 1-[(4-hydrazinobenzoyl)oxy]-monohydrochloride]

4-Hydrazinobenzoic acid, 2- (t-butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON), dicyclohexylcarbodiimide and N-hydroxysuccinimide were purchased from Aldrich Chemicals (Milwaukee, Wi.).

Synthesis of 4-BOC-Hydrazinobenzoic acid

To a stirred solution of 4-hydrazinobenzoic acid (1 equivalent) and triethylamine (3 equivalents) in dimethylformamide (5 mg/l) was added dropwise a solution of BOC-ON (1 equivalent) in dimethylformamide. The reaction mixture was stirred at room temperature for 3 hours. Ten percent aqueous hydrochloric acid was added and subsequently the solution became cloudy. The solution was extracted with ethyl acetate and the combined organic extracts were washed with water, dried over magnesium sulfate, filtered and concentrated under reduced pressure to give a brown solid. The solid was recrystallized from chloroform to give the desired product as a pale brown solid; yield 69%. Analysis:

Calculated for C12 H16 N2 O: C--57.13; H--6.39; N--11.10. Found: C--57.02; H--6.13; N--11.61.

1 H NMR δ: 1.45(s,9H), 6.77(d,Jab =8.6 Hz, 2H), 7.85(d,Jab =8.6 Hz,2H)

Synthesis of Succinimidyl 4-BOC-Hydrazinobenzoate [Hydrazinecarboxylic acid, 2-[4-[[(2,5-dioxo-1-pyrrolidinyl)oxylcarbonyl]phenyl]-1,1-dimethylethyl ester]

To a solution of 4-BOC-hydrazinobenzoic acid (1 equivalent) and N-hydroxysuccinimide (1 equivalent) in dioxane (10 ml/g of acid) was added dropwise a solution of dicyclohexylcarbodiimide (1 equivalent) in dioxane (5 ml/g). The reaction mixture was stirred at room temperature for 16 hours. Acetic acid (0.5 ml) was then added and stirring was continued for 1 hour. The reaction mixture was filtered to remove the urea byproduct. The filtrate was concentrated under reduced pressure to give a brown solid which was treated with ether and the solids were isolated by filtration to give a pale brown solid; yield 86%.

Analysis: Calculated for C16 H19 N3 O6 : C--55.01; H--5.48; N--12.03. Found: C--55.17; H--5.84; N--11.86.

1 H NMR δ: 1.47(s,9H) ,2.88(s,4H) ,6.85(d,Jab =8.9 Hz,2H) 8.04 (d,Jab =8.9 Hz,2H)

Synthesis of Succinimidyl 4-Hydrazinobenzoate Hydrochloride

To a solution of hydrogen chloride in dioxane (50 ml/g of ester; prepared by bubbling hydrogen chloride into dioxane for approximately five minutes) was added succinimidyl 4-BOC-hydrazinobenzoate (1 equivalent). The reaction mixture was stirred at room temperature. The reaction mixture was never homogeneous, however the color was initially pale brown and over 2 hours became orange. The reaction mixture was filtered and washed with ether to give a pale yellow solid; yield 72%; m.p. 203.5°-205° C.

Analysis: Calculated for C11 H12 ClN3 O4 : C--46.25; H--4.23; Cl--12.40; N--14.71; Found: C--46.74; H--4.38; Cl--12.24; N--14.26.

1 H NMR δ:2.87(s,4H),7.05(d,2H,Jab =8.9 Hz)7.97(d,2H,Jab =8.9 Hz)

EXAMPLE 2 Preparation of Succinimidyl 6-Hydrazinopyridine-3-Carboxylate Hydrochloride [2,5-Pyrrolidinedione, 1-[[6-Hydrazino-3-Pyridinyl)Carbonyl]Oxy]-, Monohydrochloride]

6-chloronicotinic acid, di-t-butyl dicarbonate and 85% hydrazine hydrate were purchased from Aldrich Chemicals (Milwaukee, Wi.).

Synthesis of 6-Hydrazinopyridine-3-Carboxylic Acid

6-Chloronicotinic acid (8.0g) was added to 85% hydrazine hydrate (35 ml). The reaction mixture was placed in a 100° C. oil bath for 4 hours. The homogeneous reaction mixture was concentrated to dryness to give a white solid. The solid was dissolved in water and on acidification to pH 5.5 with concentrated hydrochloric acid a precipitate formed. The precipitate was isolated by filtration and the solid was washed with 95% ethanol and ether to give 6.0 g of a pale brown solid; yield 77% m.p. 292°-293° C.;

Analysis: Calculated for C6 H7 N3 O2 ; C--47.06; H--4.61; N--27.44; Found: C--46.83; H--4.38; N--27.27.

1 H NMR δ: 6.69(d,J=8.8 Hz,1H),7.84(dd,J=2.4, 8.8 Hz,1H), 8.51(d,J=2.4 Hz,1H)

Synthesis of 6-BOC-Hydrazinopyridine-3-Carboxylic Acid

To a solution of 6-hydrazinopyridine-3-carboxylic acid (1.4 g; 9.8 mmol); triethylamine (1.2 ml; 11.8 mmol) in dimethylformamide (10 ml) was added di-t-butyldicarbonate (2.13 g; 9.8 mmol). The reaction mixture became homogeneous over 1 hour and stirring was continued for 16 hours at room temperature. The reaction mixture was concentrated to dryness under reduced pressure to give a brown solid. The residue was dissolved in a minimum amount of ethyl acetate and filtered through silica gel 60 (230-400 mesh) using ethyl acetate as eluent. The eluent was concentrated to dryness. The product was used without further purification.

1 H NMR δ: 1.40(s,9H),6.52(d,J=8.8 Hz,1H)7.97(dd,J=2.4, 8.8 Hz, 1H), 8.58 (d,J=2.4 Hz, 1H)

Synthesis of Succinimidyl 6-BOC-Hydrazinopyridine-3-carboxylate [Hydrazinecarboxylic acid 2-[5-[[(2,5-dioxo-1-pyrrolidinyl)oxylcarbonyl]-2-pyridinyl]--, 1,1-dimethylethyl ester]

To a solution of 6-BOC-hydrazinopyridine-3-carboxylic acid (1.45 g; 5.75 mmol) and N-hydroxysuccinimide (0.66 g; 5.75 mmol) in dimethylformamide (15 ml) was added a solution of dicyclohexylcarbodiimide (1.18 g; 5.75 mmol) in dimethylformamide (5 ml). The reaction mixture became cloudy after 1 hour and stirring was continued for 16 hours at room temperature. The reaction mixture was filtered and the filtrate was concentrated to dryness to give a brown solid residue. The residue was dissolved in a minimum amount of ethyl acetate and filtered through silica gel 60 (230-400 mesh) using ethyl acetate as eluant. The eluant was concentrated to dryness to give a pale yellow solid which was recrystallized from ethyl acetate/hexanes; yield 60%; m.p. 169.5°-172° C.;

Analysis: Calculated for C15 H18 N4 O6 ; C--51.43; H--5.18; N--15.99 Found: C--51.81; H--5.26; N--15.60.

1 H NMR δ: 1.41(s,9H),2.87(s,4H)6.64(d,J=8.8 Hz,1H)8.08 (dd,J=2.4, 8.8 Hz) 8.73 (d,J=2.4 Hz, 1H)

Synthesis of Succinimidyl 6-Hydrazinopyridine-3-Carboxylate Hydrochloride

A solution of hydrogen chloride in dioxane was prepared by bubbling anhydrous hydrogen chloride into dioxane (20 ml) at a moderate rate for 10 min. Succinimidyl 6-BOC-hydrazinopyridine-3-carboxylate (100 mg) was dissolved in dioxane (2 ml) and HCl/dioxane (2 ml) was added and the reaction mixture was stirred at room temperature. After 5 minutes the solution became cloudy and a precipitate formed. Stirring was continued for 4 hours. The cloudy reaction mixture was filtered to give 55 mg of a white solid; yield 67%;

Analysis: Calculated for C10 H11 ClN4 O4 ; C--41.87; H--3.87; Cl--12.37; N--19.53; Found: C--41.92; H--3.90; Cl--12.30; N--19.47.

1 H NMR δ: 2.88(s,4H),7.01(d,J=8.8 Hz,1H)8.19(dd,J=2.4, 8.8 Hz, 1H) 8.83 (d,J=2.4 Hz, 1H)

EXAMPLE 3 Preparation of Succinimidyl 4-Hydrazidodoterephthalate Hydrochloride [Benzoic Acid, 4-[[(2,5-Dioxo-1-Pyrrolidinyl) Oxy]carbonyl]--, Hydrazide, Monohydrochloride]

Mono-methyl terephthalate, oxalyl chloride, t-butyl carbazate, dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were purchased from Aldrich Chemicals (Milwaukee, Wi.).

Synthesis of Methyl Terephthalate Chloride

To a stirred solution of mono-methyl terephthalate (1 equivalent), toluene (30 ml/gm of ester) and 3 drops of DMF was added dropwise oxalyl chloride (2.0 equivalents). The reaction mixture was stirred at 45° C. for 16 hours. The solution was concentrated under reduced pressure to give the desired product as a pale yellow solid. The product was used without further purification; yield 82.0%; m.p. 50°-52° C. IR (thin film); 2970, 1775, 1720, 1430, 1400, 1280, 1105, 880 cm-1.

1 H NMR δ: 3.97(s,3H),8.14(s,4H).

Synthesis of Methyl 4-BOC-Hydrazidoterephthalate [1,4-Benzenedicarboxylic Acid, Monomethyl Ester, 2-[(1,1-Dimethylethoxy) Carbonyl]Hydrazide]

To a vigorously stirred mixture of t-butyl carbazate (1 equivalent), methylene chloride (20 ml/gm) and 25% sodium bicarbonate (2.0 equivalents) was added dropwise a solution of methyl terephthalate chloride (1 equivalent) in methylene chloride (40 ml/gm). The reaction mixture was stirred at 20° for 1/2 hour. The phases were separated and the aqueous phase was extracted with methylene chloride. The combined organic phases were washed with 10% hydrochloric acid and brine. The organic phase dried (MgSO4), filtered and concentrated to give a white solid; yield 91.7%; m.p. 197°-199°. IR (KBr): 3010, 1720, 1670, 1430, 1270, 1220, 1130, 1100, 1030, 870, 750 cm-1.

1 H NMR(CDCl3): δ1.49(s,9H),3.93(s,3H),7.83(d,JAB =8 Hz,2H),8.07 (d,JAB =8 Hz ,2H).

Synthesis of 4-BOC-Hydrazidoterephthalic Acid

To a solution of methyl 4-BOC-hydrazidoterephthalate (1 equivalent) in methanol (50 ml/gm) was added sodium hydroxide (10.0 equivalents). The reaction was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure to remove the methanol. Water was added and the solution was carefully acidified to pH 1.0. The acidic solution was extracted with ethyl acetate and the organic extract was dried (MgSO4), filtered and concentrated to dryness under reduced pressure to give a white solid; yield 87.5%; m.p. 208°-210°.

1 H NMR δ: 1.41 (s,9H),7.97(d,J=2.4 Hz,4H),8.90(m, 1H) 10.3(m, 1H).

Synthesis of Succinimidyl 4-BOC-Hydrazidoterephthalate ]Hydrazinecarboxylic Acid, 2-[4-[[(2,5-dioxo-1-Pyrrolidinyl)Oxy]Carbonlyl]Benzoyl]--, 1,1-dimethylethyl ester]

To a solution of 4-BOC-hydrazidoterephthalic acid (1 equivalent) and N-hydroxysuccinimide (1 equivalent) in DMF (10 ml/gm) was added dropwise a solution of DCC (1 equivalent) in DMF (5 ml/gm). The reaction mixture was stirred at 20° for 16 hours. Acetic acid (0.5 ml) was then added and stirring was continued for 1 hour. The reaction mixture was filtered to remove the urea byproduct. The filtrate was concentrated under reduced pressure to give a yellow brown oil. Flash vacuum chromatography (hexanes/ethyl acetate (7/3)) was used to isolate the product; yield 47.8%, m.p. 182°-185°. IR (KBr): 3330, 3230, 2990, 1770, 1740, 1660, 1530, 1500, 1370, 1280, 1200, 1150, 1070, 1000, 870, 790, 640 cm-1.

1 H NMR (CDCl3) δ: 1.50(s,9H),2.91(s,4H),6.70(m, 1H),7.91(d,JAB =8.8 Hz,2H), 8.20(d, JAB =8.8 Hz, 2H).

Analysis: Calculated for C17 H19 N3 O7 : C--54.11; H--5.07; N--11.13; Found: C--53.66; H--5.15; N--11.09.

Synthesis of Succinimidyl-4-Hydrazidoterephthalate Hydrochloride

To a solution of hydrogen chloride in tetrahydrofuran (50 ml/gm; prepared by bubbling hydrogen chloride into tetrahydrofuran for approximately ten minutes) was added succinimidyl 4-BOC-hydrazidoterephthalate (1 equivalent). The reaction mixture was homogeneous for 1 hour; then over 4 hours a pale white precipitate formed. The reaction mixture was filtered and washed with ether to give the desired product as a pale white solid; yield 37.7%; m.p. 278°-280°. IR(KBr): 3400, 3200, 2800, 2600, 1770, 1730, 1690, 1530, 1490, 1290, 1240, 1070, 1000, 870, 730, 640, 610 cm-1.

1 H NMR δ: 2.91 (s,4H),8.11(d,JAB =8.8 Hz,2H),8.25(d,JAB =8.8 Hz,2H).

Analysis: Calculated for C12 H12 ClN3 O5 : C--45.95; H--3.86; Cl--11.30; N--13.40; Found: C--45.84; H--3.91; Cl--11.37; N--13.33.

EXAMPLE 4 Preparation of 5-Maleimidyl-2-Hydrazinopyridine Hydrochloride [1H-Pyrrole-2,5-dione, 1-(6-Hydrazino-3-Pyridinyl)--, Monohydrochloride]

2-chloro-5-nitropyridine, hydrazine hydrate, di-tert-butyl dicarbonate, 10% palladium on charcoal, maleic anhydride, cobalt acetate and acetic anhydride were purchased from Aldrich Chemicals (Milwaukee, Wi.).

Synthesis of 2-Hydrazino-5-Nitropyridine

To a stirred solution of hydrazine hydrate (30.0 equivalents), water (4 ml/gm of pyridine), and ethanol (2 ml/gm of pyridine) was added 2-chloro-5-nitropyridine (1 equivalent). The reaction mixture was stirred at 20° for 16 hours (a very thick green slurry forms). The precipitate was isolated by filtration and the solid was washed with methanol and then ether to give a green solid. The product was used without further purification; yield 77.3%; m.p. 205°-207°. IR {KBr): 3340, 3200, 2980, 1670, 1605, 1580, 1485, 1420, 1330, 1300, 1120, 980, 830, 770 cm-1.

1 H NMR δ:4.64(br s,2H),6.76{d,J=8.8 Hz,1H),8.15(dd,J=2.4, 8.8 Hz,1H), 8.86(d,J=2.4 Hz,1H),9.12,(m,1H).

Analysis: Calculated for C5 H6 N4 O2 : C--39.21; H--3.93; N--36.51; Found: C--38.96; H--3.92; N--36.35.

Synthesis of 2-(BOC-Hydrazino)-5-Nitropyridine

To a stirred solution of 2-hydrazino-5-nitropyridine (1 equivalent), DMF (15 ml/gm of pyridine), and triethylamine (1.1 equivalents) was added dropwise a solution of di-tert-butyl dicarbonate (1.0 equivalent) in DMF (4 ml/gm of dicarbonate). The reaction mixture was stirred at 20° for 48 hours. The reaction mixture was concentrated under reduced pressure to a yellow brown oil. Flash vacuum chromatography (hexanes/ethyl acetate (8/2)) was used to isolate the product. The product was recrystallized from ethyl acetate/hexanes; yield 63.4%; m.p. 135°-137°. IR (KBr): 3280, 2980, 1710, 1600, 1500, 1330, 1290, 1270, 1250, 1150, 1120, 1010, 830, 760, 650, 500 cm-1.

1 H NMR δ: 1.41(s,9H),6.60(d,JAB =8.8 Hz,1H),8.28(dd,J=2.4,8.8 Hz,1H), 8.93(d,JAB =2.4 Hz, 1H),9.14(m, 1H),9.56(m,1H).

Analysis: Calculated for C10 H14 N4 O4 : C--47.24; H--5.55; N--22.03; Found: C--46.99; H--5.50; N--21.93.

Synthesis of 2-(BOC-Hydrazino)-5-Amino-Pyridine

Into a Parr hydrogenation bottle was added 2-BOC-hydrazino-5-nitropyridine (1 equivalent), 1(7% palladium on charcoal (0.3 gm Pd/gm of pyridine) and ethanol (100 ml/gm of pyridine). The reaction was hydrogenated at 50 psi H2 for 2 hours at room temperature on a Parr hydrogenator. The reaction mixture was filtered through a filter cell plug and rinsed with ethanol. The yellow green solution was concentrated under reduced pressure to give a pale yellow solid. The product was recrystallized from ethanol; yield 81.64%; m.p. 140°-142°. IR (KBr): 3360, 3200, 2980, 1650, 1635, 1580, 1490, 1390, 1360, 1300, 1260, 1160, 1020, 880, 860, 830, 750, 590, 530 cm-1.

1 H NMR δ: 1.37(s,9H),6.34(d,J=8.8 Hz,1H),6.89(dd,J=2.4,8.8 Hz,1H), 7.14(m, 1H), 7.49(d,J=2.4 Hz, 1H),8.50(m, 1H).

Analysis: Calculated for C10 H16 N4 O2 : C--53.55; H--7.19; N--24.98; Found: C--53.73; H--7.21; N--25.05.

Synthesis of 2-BOC-Hydrazino-5-Maleimidylpyridine [Hydrazinecarboxylic Acid, 2-[5-(2,5-Dihydro-2,5-dioxo-1H-Pyrrol-1Yl)-2-Pyridinyl]--, 1,1-Dimethylethyl Ester]

To a stirred solution of 2-BOC-hydrazino-5-amino pyridine (1 equivalent) in acetone (50 ml/gm) was added maleic anhydride (1.1 equivalents). The reaction mixture was stirred at 25° for 2 hours. To the reaction mixture was added acetic anhydride (1.2 equivalents), cobalt acetate (0.007 equivalents) and triethylamine (0.3 equivalents). The reaction mixture was stirred at 60° for 2 hours. The color of the reaction began as bright yellow and ended as dark yellow. The reaction mixture was concentrated under reduced pressure to give a yellow brown oil. Flash vacuum chromatography (hexanes/ethyl acetate (8/2)) was used to isolate the product. The product was recrystallized in ether/hexane; yield 28.3%; m.p. 182°-184°. IR (KBr): 3400, 3300, 3100, 2990, 1700, 1610, 1500, 1410, 1360, 1320, 1270, 1210, 1150, 1050, 830, 750, 690 cm-1.

1 H NMR δ:1.39(s,9H),6.S8(d,J=8.8 Hz,1H),7.14(s,2H),7.45 (dd,J=2.4,8.8 Hz,]H),7.94(d,J:2.4 Hz,1H),8.37(m, 1H).

Analysis: Calculated for C14 H16 N4 O4 : C--55.26; H--5.30; N--18.41; Found: C--55.14; H--5.30; N--18.33.

Synthesis of 5-Maleimidy]-2-Hydrazinopyridine Hydrochloride

A solution of hydrogen chloride in dioxane was prepared by bubbling anhydrous hydrogen chloride into dioxane (50 ml) at a moderate rate for 10 minutes. 2-(BOC-hydrazino)-5-maleimidylpyridine (200 mg) was dissolved in dioxane (5 ml) and HCl/dioxane (10 ml) was added and the reaction mixture was stirred at room temperature. After 30 minutes the solution became cloudy and a precipitate formed. The reaction mixture was stirred at 25° for a total of 5 hours. The slurry was filtered and washed with ether to give 50 mg of a white solid; yield 31.6%; m.p. 280°-290° (decomp.; yellow to brown). IR (KBr): 3440, 3100, 2580, 1720, 1610, 1560, 1480, 1390, 1200, 1150, 830, 690 cm-1.

1 H NMR δ:7.0(d,J=8.8 Hz,1H),7.19(s,2H),7.63(dd,J-2.4,8.8 Hz,1H), 8.15(d,J=2.4 Hz,1H). Mass spectrum: m/z=204 (M-HCl)+.

EXAMPLE 5 Preparation of Succinimidyl 2-(2-Propenylhydrazone)Nicotinate [Propanal, [5-[[(2,5-Dioxo-1-Pyrrolidinyl)Oxy]Carbonyl]-2-Pyridinyl]Hydrazone]

6-Hydrazinonicotinic acid was prepared as previously described, and propionaldehyde was purchased from Aldrich Chemicals {Milwaukee, Wi.).

Synthesis of Succinimidyl 2-(2-Propenylhydrazone)Nicotinate

To a suspension of 6-hydrazinonicotinic acid (1 equivalent) in DMF (40 ml/g) was added propionaldehyde (3 equivalents). The reaction mixture was stirred at ambient temperature for 1 hour. If the reaction mixture did not become homogeneous the flask was gently heated with a heat gun until the reaction mixture became homogeneous. The reaction mixture was cooled to ambient temperature and a solution of N-hydroxysuccinimide (1 equivalent) in DMF was added. Subsequently a solution of DCC (1 equivalent) in DMF was added dropwise. The reaction mixture was stirred for 16 hours at ambient temperature. The precipitate which formed was removed by suction filtration and the mother liquors were concentrated to dryness. The brown solid residue was suspended in ethyl acetate and stirred for 1 hour and filtered. A pale brown solid precipitated from the ethyl acetate solution and was isolated by filtration to give the desired product; yield 65%.

1 H NMR δ: 1.06(t,3H),2.34(m,2H),2.86(s,4H),7.11(d,J=9.4 Hz,1H),7.54

(t,1H,J=4.9 Hz),8.10(dd,J=9.44,2.33 Hz,1H),8.72(d,J=2.33 Hz,1H) Analysis: Calculated for C13 H14 N4 O4 : C--53.79; H--4.86; N--19.30; Found: C--53.66; H--4.89; N--19.12.

EXAMPLE 6 Preparation of Succinimidyl 2-(2-(1-Propenyl)Hydrazone))-Thiazole-4-Carboxylate [Propanal, [4-[[(2,5-Dioxo-1-Pyrrolidinyl)Oxy]Carbonyl]-2-Thiazolyl]Hydrazone]

Thiosemicarbazide and bromolactic acid were purchased from Aldrich Chemicals (Milwaukee, Wi.).

Synthesis of Propionaldehyde Thiosemicarbazone

To a solution of thiosemicarbazide (1 equivalent) and propionaldehyde (1.5 equivalents) in MeOH was added a few drops of glacial acetic acid. The mixture was heated to reflux for 45 minutes. The reaction mixture was concentrated on the rotovap which caused a precipitate to form. The solids were isolated by filtration, washed with ether and dried in vacuo; yield 71%.

Synthesis of 2-(2-(1Propenylhydrazone))Thiazole-4-Carboxylic Acid Hydrobromide

To a solution of propionaldehyde thiosemicarbazone (1 equivalent) in MeOH was added bromolactic acid (1 equivalent). The reaction mixture was heated at reflux for 1 hour, then cooled to room temperature and the solvent was removed on the rotavap. The resulting yellow solid was triturated with MeOH/ether and a pale yellow solid was isolated and washed with ether and dried in vacuo.

1 H NMR δ:1.00(t,J=7.9 Hz,3H),2.15(m,2H),7.44(t,J=4.9 Hz).

Syntheses of Succinimidyl 2-(2-(1-Propenyl)Hydrazone)-Thiazol-4-carboxylate

To a solution of acid (1 equivalent), N-hydroxysuccinimide (1 equivalent) and triethylamine (1.5 equivalents) in DMF was added dropwise a solution of DCC (1 equivalent) in DMF. The reaction mixture was stirred for 16 hours at room temperature. The precipitate which formed was removed by filtration and the mother liquors were concentrated to dryness. Ethyl acetate was added to the residue and stirred for 1 hour. The insolubles were removed by filtration and the mother liquor was concentrated to dryness. The product was flash chromatographed using hexanes/ethyl acetate (1/2) as eluant to give the desired product as an off-white solid; yield 35%.

1 H NMR δ:1.08(t,J=7.9 Hz,3H),2.24(m,2H),7.40(t,J=4.9 Hz,1H),8.11 (s,1H).

EXAMPLE 7 Preparation of Succinimidyl 2-(2-Methylethenylhydrazone) Thiazole-4-Carboxylate Synthesis of Succinimidyl 2(2-Methylethenylhydrazone) Thiazole-4-Carboxylate

2-(Methylethyenylhydrazone)-4-Thiazolecarboxylic Acid hydrobromide (1 equivalent) (prepared according to the method of H. Johne, D. Selferr, S. Johne and E. Bulka, Pharmazie 33, 259 (1978)) was dissolved in DMF (20 ml/g). N-Hydroxysuccinimide (1 equivalent) and triethylamine equivalents) were added. To the homogeneous mixture a solution of DCC (1 (1.5 equivalent) was added dropwise over 15 minutes. The reaction mixture was stirred for 16 hours at room temperature. The precipitate which formed was removed by filtration and the mother liquor was concentrated to dryness to give an orange-brown solid. The residue was suspended in ethyl acetate and stirred at room temperature for 1 hour. Insolubles were removed by filtration and the mother liquor was concentrated to give a brown solid. The solids were triturated with ether and re-isolated by filtration to give a yellow-brown solid: yield 40%. A sample of the product was filtered through a short plug of silica gel using hexanes/ethyl acetate (2/1) as eluate. The eluant was concentrated to give the desired product as a pale yellow solid; m.p. 202°-205° (decomp.).

1 H NMR δ: 1.92(s,3H),1.96(s,3H),2.86(s,4H),8.11(s,1H). Mass spectrum: m/z=297 (M+1)+

EXAMPLE 8 Preparation of Succinimidyl 4-Thiosemicarbazidobenzoate Hemihydrochloride [Hydrazinecarbothioamide, N-[4-[[(2,5-Dioxo-1-Pyrrolidinyl)Oxyl]Carbonyl]Phenyl]--, Hemihydrochloride].

4-Aminocarboxylic acid was purchased from Aldrich Chemicals (Milwaukee, Wi.).

Synthesis of BOC-4-Thiosemicarbazidobenzoic Acid [Hydrazinecarboxylic Acid, 2-[[(4-carboxyphenyl)Amino)Thioxomethyl]--, 1-(1,1-dimethylethyl) Ester]

To a solution of 4-isothiocyanatobenzoic acid (1 equivalent) (prepared according to the method of D. W. Browne and G. M. Dyson, J. Chem. Soc. 178 (1934)) and triethylamine (1.2 equivalents) in DMF was added to a solution of t-butyl carbazate (1 equivalent). The reaction mixture was stirred at room temperature for 3 hours and subsequently concentrated to dryness. The residue was dissolved in ethyl acetate and washed with 10% citric acid and brine. The organic phase was dried (MgSO4), filtered and concentrated to give the desired product as an off-white solid; yield 70%; m.p. 131°-133° (decomp.).

1 H NMR δ: 1.42(s,9H),7.67(d,JAB =8.9 Hz,2H),7.87(d,JAB =8.9 Hz,2H).

Synthesis of Succintmidyl BOC-4-Thiosemicarbazidobenzoate [Hydrazinecarboxylic Acid, 2-[[[4-[[(2,5-dioxo-1-Pyrrolidinyl)oxyl]Carbonyl]Phenyl]Amino]Thioxomethyl]-1,1-Dimethylethyl Ester]

To a solution of acid (1 equivalent) and N-methylmorpholine (1.1 equivalents) in acetonitrile was added a solution of succinimidyl tetrachloroethyl carbonate (1 equivalent) in acetonitrile. The reaction mixture was stirred at room temperature for 16 hours. Ethyl acetate was added to the reaction mixture and the homogeneous solution was washed with cold 5% citric acid, cold water, cold aqueous saturated sodium bicarbonate solution, cold water and cold brine. The organic phase was dried (MgSO4), filtered and concentrated to give a pale yellow oil. The oil was flash chromatographed on silica gel using hexanes/ethyl acetate as eluant. The product was isolated as a yellow oil which solidified on the addition of ether. The solids were isolated by filtration to give the desired product; yield 25%; m.p. 161°-163°.

1 H NMR δ: 1.52(s,9H),2.88(s,4H),7.74(d,JAB =10.0 Hz,2H), 8.05(d,JAB =10.0 Hz, 2H).

Analysis: Calculated for C17 H20 N4 SO6 : C--49.99; H--4.94; N--13.72 S--7.85; Found: C--50.05; H--4.95; N--13.64 s--7.95.

Synthesis of Succinimidyl 4-Thiosemicarbazidobenzoate Hemihydrochloride

To a suspension of BOC succinimidyl ester in ether was added a solution of dry HCl (g) in ether (prepared by bubbling HCl gas into dry ether). The suspension was stirred at room temperature for 16 hours. The reaction mixture was heterogeneous over the entire course of the reaction. The solids were isolated by filtration to give the desired hydrochloride salt product; yield 80%; m.p. 155°-160°.

1 H NMR δ: 2.88(s,4H), 8.01(s,4H).

Analysis: Calculated for C12 H13 N4 SO4.0.5 HCl: C--44.00; H--4.15; N--17.10 S--9.79; Found: C--44.56; H--3.88; N--17.04 S--9.74.

EXAMPLE 9 Conjugation of IgG

To a solution of 10 mg IgG (MW=155,000) in 2 ml 0.1M sodium phosphate buffer (pH 7.8) was added 17.2 μl of 30 mM succinimidyl 4-hydrazinobenzoate hydrochloride in dimethylformamide. After stirring for 5 hours at room temperature, the reaction mixture was dialyzed against 0.1M sodium acetate buffer (pH 5.2). The number of hydrazino groups conjugated onto the protein was measured by the method of T. P. King et al. (Biochemistry, 25:5774, 1986). Briefly, the hydrazino-protein conjugate was reacted with 4-nitrobenzaldehyde to convert the hydrazino groups into hydrazones. The number of hydrazones/protein molecule were determined spectrophotometrically using the hydrazone of p-nitrophenylbenzaldehyde and phenylhydrazine (λmax =412, ε=2.41×104) as a standard. Modification yields of 25-35% were obtained.

EXAMPLE 10 Labelling of Conjugated IgG With 99m Tc

A DuPont Tc-glucoscan kit was reconstituted with 3 ml of water containing 10 mCi of 99m TcO4 -. 250 μl of this solution was mixed with 250 μl of 1-5 mg/ml conjugated IgG in 0.1M sodium acetate buffer (pH 5.2). After incubation for 1 hour at room temperature >95% of the activity was associated with the protein as determined by radiometric HPLC (TSK 3000 column) and instant thin layer chromatography (ITLC.)

800 uCi of Tc labelled IgG was injected into rats containing an abscess in one hind leg. At 24 hours the rats were sacrificed. The distribution of radioactivity was measured:

______________________________________          % InjectedOrgan          dose/gram tissue______________________________________Blood          1.36Kidney         1.11Infected Muscle          0.76Normal Muscle  0.12Liver          0.81______________________________________ Ratio Infected/Normal muscle = 6.3
EXAMPLE 11 Conjugation of Fragment E1

(DD)E protein was concentrated to between 5-10 mg/ml and modified with a 20-fold molar excess of succinimidyl 6-hydrazinopyridine-3-carboxylate hydrochloride in 12.5 mM borate buffer at pH 8.5. After a 5 hour incubation (with gentle stirring) at 4° C., the sample was dialyzed approximately 12 hours versus degassed nanopure water.

Fragment E1 was separated from the modified DD(E) complex by making the sample 0.55M in acetic acid and diluting 1:1V/V with 6M urea. The pH of the sample was adjusted to 5.5 with 10N NaOH and the sample was then dialyzed against 10 mM citrate buffer (pH 5.7) to remove excess reagents. During dialysis, DD protein precipitates leaving modified fragment E1 in solution. The DD precipitate was readily removed by centrifugation.

Modified E1 was labelled with Tc-99m via reaction with Tc-99m glucoheptonate, as previously described. The Tc-ggm labelled E1 was used to image a thrombus in a rabbit model {see D. Collen et al., J. Clin. Invest. 71, p. 368-376 (1983)).

EXAMPLE 12 Conjugation of Monoclonal Antibody 5E8

(see E. A. Chen et al, Cancer Research, 49, p. 3642-3649 (1989)).

5E8 (at a concentration of 5-10 mg/ml) was modified with a 14-fold excess of succinimidyl 6-hydrazinopyridine-3-carboxylate hydrochloride in 12.5 mM borate buffer at pH 8.5 (5 hours at room temperature). The antibody conjugate was dialyzed against a 20 mM citrate buffer (pH 5.2 100 mM in NaCl). After centrifugation to remove a small amount of turbidity, the degree of modification (determined spectrophotometrically, as previously described) was found to be 6.5 hydrazine groups per protein molecule. Analysis by ELISA and immunocytoadherence showed no loss in immunoreactivity.

The specific compounds and details of the method described above are exemplary and are not intended to limit the scope of the invention.

Claims (17)

We claim:
1. A hydrazine or hydrazide compound of the formula (I) or (II): ##STR5## wherein: A is a carbon or nitrogen atom;
B is a carbon or nitrogen atom;
D is a direct bond, ##STR6## --CH2, or ##STR7## E is C═O and F is an N-oxysuccinimidyl, tetrafluorophenolate, N-oxybenztriazole, or imidazolate group, or E and F together form a maleimidyl group;
R is hydrogen or a lower alkyl group;
R' and R" may be the same or different and are selected from hydrogen and lower alkyl; and
X is a negative counterion.
2. The compound of claim 1 wherein F is ##STR8##
3. A hydrazine or hydrazide compound of the formula (I) or (II): ##STR9## wherein: A is a carbon or nitrogen atom;
B is a carbon or nitrogen atom;
D is a direct bond, CH2, C═O or ##STR10## E is C═O; F is N-oxysuccinimidyl;
R is hydrogen or methyl;
R' and R" may be the same or different and are selected from hydrogen and lower alkyl; and
X is Cl.
4. A hydrazine or hydrazide compound of the formula (I) or (II): ##STR11## wherein: A is a carbon or nitrogen atom;
B is a carbon or nitrogen atom;
D is a direct bond, CH2, C═O or thioamide ##STR12## attached to the 4-position of the ring; E is C═O and
F is an N-oxysuccinimidyl, tetrafluorophenolate, N-oxybenztriazole, or imidazolate group, or E and F together form a maleimidyl group;
R is hydrogen or a lower alkyl group;
R' and R" may be the same or different and are selected from hydrogen and lower alkyl; and
X is a negative counterion.
5. The compound of claim 1 wherein D is a direct bond to the 4-position of the ring.
6. The compound of claim 5 wherein E is carbonyl and F is selected from the group consisting of N-oxysuccinimidyl, tetrafluorophenolate, N-oxybenztriazole and imidazolate, and X is selected from the group consisting of halides, nitrate, trifluoroacetate, tetrafluoroborate and sulfate.
7. The compound of claim 5 wherein R is hydrogen or methyl, E is carbonyl, F is N-oxysuccinimidyl, and X is Cl.
8. The compound of claim 7 wherein both A and B are carbon atoms or one of A and B is carbon and the other is nitrogen.
9. The compound of claim 7 wherein A is carbon, B is nitrogen, and R is hydrogen.
10. The compound of claim 4 wherein D is C═O or thioamide.
11. The compound of claim 10 wherein E is C═O and F is selected from the group consisting of N-oxysuccinimidyl, tetrafluorophenolate, N-oxybenztriazole and imidazolate and X is selected from the group consisting of halides, nitrate, trifluoroacetate, tetrafluoroborate and sulfate.
12. The compound of claim 4 wherein D and E are carbonyl, F is N-oxysuccinimidyl and X is Cl.
13. The compound of claim 4 wherein D is thioamide, E is carbonyl, F is N-oxysuccinimidyl and X is Cl.
14. The compound of claim 5 wherein E is carbonyl, F is N-oxysuccinimidyl, A is carbon, B is nitrogen, R and R' are hydrogen, and R" is ethyl.
15. A hydrazine compound of the type: ##STR13## where E is C═O and F is an oxysuccinimidyl, tetrafluorophenolate, N-oxybenztriazole, or imidazolate group or E and F together form a maleimidyl group; R, R' and R" are the same or different and are selected from the group consisting of hydrogen and lower alkyl.
16. A compound of claim 15 wherein E is carbonyl, F is N-oxysuccinimidyl, R' is hydrogen and R" is ethyl.
17. A compound of claim 16 wherein E is carbonyl, F is N-oxysuccinimidyl, and both R' and R" are methyl.
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070685A3 (en) * 2000-03-22 2003-03-27 Solulink Inc Hydrazine-based and carbonyl-based bifunctional crosslinking reagents
US20030125707A1 (en) * 2001-12-31 2003-07-03 Kimberly-Clark Worldwide, Inc. Mechanical fastening system for an absorbent article
US6686461B1 (en) 2000-03-22 2004-02-03 Solulink Bioscience, Inc. Triphosphate oligonucleotide modification reagents and uses thereof
US20050099738A1 (en) * 2003-11-06 2005-05-12 Seagate Technology Llc Magnetoresistive sensor having specular sidewall layers
US20050203289A1 (en) * 2000-03-22 2005-09-15 Schwartz David A. Functional biopolymer modification reagents and uses thereof
US20060228348A1 (en) * 2005-04-06 2006-10-12 Genzyme Corporation Targeting of glycoprotein therapeutics
US20070080228A1 (en) * 2000-11-24 2007-04-12 Knowles C H Compact bar code symbol reading system employing a complex of coplanar illumination and imaging stations for omni-directional imaging of objects within a 3D imaging volume
US20080221343A1 (en) * 2006-04-18 2008-09-11 Schwartz David A Novel hydrazone-based and oxime-based fluorescent and chromophoric/pro-fluorescent and pro-chromophoric reagents and linkers
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Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5066789A (en) * 1988-09-30 1991-11-19 Neorx Corporation Targeting substance-diagnostic/therapeutic agent conjugates having Schiff base linkages
US6001979A (en) 1989-08-29 1999-12-14 Nycomed Amersham Plc Cores for technetium radiopharmaceuticals
US5750088A (en) 1993-03-30 1998-05-12 The Dupont Merck Pharmaceutical Company Stable hydrazones linked to a peptide moiety as reagents for the preparation of radiopharmaceuticals
US5449761A (en) * 1993-09-28 1995-09-12 Cytogen Corporation Metal-binding targeted polypeptide constructs
US5879659A (en) * 1996-03-13 1999-03-09 Dupont Pharmaceuticals Company Ternary radiopharmaceutical complexes
US6403054B1 (en) 1997-05-28 2002-06-11 Bristol-Myers Squibb Pharma Company Ternary ligand complexes useful as radiopharmaceuticals
US6251364B1 (en) 1999-03-29 2001-06-26 Dupont Pharmaceuticals Company Ternary ligand complexes useful as radiopharmaceuticals
US6534038B2 (en) 2000-04-07 2003-03-18 Bristol-Myers Squibb Pharma Company Ternary ligand complexes useful as radiopharmaceuticals
WO2002055112A2 (en) * 2001-01-09 2002-07-18 Squibb Bristol Myers Co Polypodal chelants for metallopharmaceuticals
US6776977B2 (en) * 2001-01-09 2004-08-17 Bristol-Myers Squibb Pharma Company Polypodal chelants for metallopharmaceuticals
US6517814B2 (en) 2001-01-09 2003-02-11 Bristol-Myers Squibb Pharma Company Macrocyclic chelants useful for metallopharmaceuticals
US7317104B2 (en) 2003-06-13 2008-01-08 Bristol-Myers Squibb Pharma Company Chelants and macrocyclic metal complex radiopharmaceuticals thereof
US7319149B2 (en) * 2003-06-13 2008-01-15 Bristol-Myers Squibb Pharma Company Chelants and macrocyclic metal complex radiopharmaceuticals thereof
WO2008121836A1 (en) * 2007-03-30 2008-10-09 Brigham And Women's Hospital, Inc. Compounds and methods for enhancing mhc class ii therapies
JP6415979B2 (en) 2011-06-03 2018-10-31 スリーエム イノベイティブ プロパティズ カンパニー Hydrazino 1h- imidazoquinoline-4-amine and conjugates prepared from this
CA2838158A1 (en) 2011-06-03 2012-12-06 3M Innovative Properties Company Heterobifunctional linkers with polyethylene glycol segments and immune response modifier conjugates made therefrom

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2101118A (en) * 1981-04-30 1983-01-12 Ikeda Mohando Co Technetium (99m)-labeled compound and process for production of said compound
EP0188256A2 (en) * 1985-01-14 1986-07-23 NeoRx Metal radionuclide labeled proteins for diagnosis and therapy
AU8226387A (en) * 1986-12-10 1988-06-16 Cis Bio International A process for the preparation of an organ-specific substance labeled with technetium-99m
US4861869A (en) * 1986-05-29 1989-08-29 Mallinckrodt, Inc. Coupling agents for joining radionuclide metal ions with biologically useful proteins

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5514676A (en) * 1984-03-19 1996-05-07 The Rockefeller University Amino-benzoic acids and derivatives, and methods of use
US4749647A (en) * 1984-06-22 1988-06-07 Genetic Systems Corporation Polymerization-induced separation assay using recognition pairs

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2101118A (en) * 1981-04-30 1983-01-12 Ikeda Mohando Co Technetium (99m)-labeled compound and process for production of said compound
EP0188256A2 (en) * 1985-01-14 1986-07-23 NeoRx Metal radionuclide labeled proteins for diagnosis and therapy
US4861869A (en) * 1986-05-29 1989-08-29 Mallinckrodt, Inc. Coupling agents for joining radionuclide metal ions with biologically useful proteins
AU8226387A (en) * 1986-12-10 1988-06-16 Cis Bio International A process for the preparation of an organ-specific substance labeled with technetium-99m

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
Arano et al, Appl. Radiat. Isot., 37(7):587 592 (1986). *
Arano et al, Appl. Radiat. Isot., 37(7):587-592 (1986).
Arano et al., J. of Nuclear Medicine, 28(6):1027 1033 (Jun. 1987). *
Arano et al., J. of Nuclear Medicine, 28(6):1027-1033 (Jun. 1987).
Chemical Abstracts, vol. 101 (No. 11) Abst. No. 101 90524 e Sep. 10, 1984. *
Chemical Abstracts, vol. 101 (No. 11) Abst. No. 101-90524-e Sep. 10, 1984.
Chemical Abstracts, vol. 106 (No. 5) Abst. No. 106:33003p Feb. 2, 1987. *
Chemical Abstracts, vol. 67 (No. 7) Abst. No. 31,163t Aug. 14, 1967. *
Chemical Abstracts, vol. 85 (No. 9) Abst. No. 85:63001(b) Aug. 30, 1976. *
Eckelman et al, Nucl. Med. Biol, 16(2):171 176 (1989). *
Eckelman et al, Nucl. Med. Biol, 16(2):171-176 (1989).
King et al. Biochemistry, 25:5774 5779 (1986). *
King et al. Biochemistry, 25:5774-5779 (1986).
Lanteigne et al., Int. J. Appl. Radiat. Isot., 35(7):617 621 (1984). *
Lanteigne et al., Int. J. Appl. Radiat. Isot., 35(7):617-621 (1984).

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